Human monocytotropic ehrlichiosis, an emerging tick transmitted disease, is the most prevalent life-threatening rickettsiosis in America, yet no vaccine is available. The long term goal of this project is to develop vaccines that provide protection against antigenically and genetically diverse ehrlichiae. The objective of this application is to identify the protective T cell antigens and immune mechanisms that confer cross-protection against antigenically and genetically diverse Ehrlichia species. We propose to exploit new mouse models of cross- and homologous protection against a highly virulent toxic shock-like monocytotropic ehrlichiosis to identify immunodominant cross-protective T cell antigens that will provide broad protection against diverse Ehrlichia strains. The specific aims are to 1) determine the specificity and protective ability of cross-reactive Ehrlichia-specific T cells to antigenically diverse Ehrlichia, 2) identify E. muris antigens recognized by Ehrlichia-specific cross-reactive T cells, and 3) determine the in vivo and ex vivo responses to optimized subunit vaccines delivered by gene and viral vectors and compare them to those associated with E. muris infection. We will determine at a clonal level the cross-reactivity of E. muris memory T cells with the Ehrlichia species isolated from Ixodes ovatus ticks (IOE) that causes fatal ehrlichiosis in immunocompetent mice. The cross-reactive T cells will be cloned, characterized for their antigen specificity, CD4/CD8 subtypes, cytokine profile, and CTL activity, and evaluated for ability to protect against fatal IOE infection. Both T-cell screening of a genomic library and proteomic approaches will be utilized to identify E. muris antigens recognized by the protective T cell clones, including secreted and cell-associated antigens, expressed at high or low levels, particularly those that are conserved. We will further determine whether an ehrlichial vaccine employing plasmid DNA delivered with Th1-promoting IL-12 or utilizing a recombinant replication-deficient adenoviral vector is more useful as a broadly effective vaccine that protects animals against lethal IOE infection, significantly decreases the bacterial load, and stimulates the established protective mechanisms of Ehrlichia-specific CD4 Th1 effector and long term memory responses, CDS CTL activity, and IgG2a antibodies. The identification of conserved immunodominant Ehrlichia antigens that are protective in the IOE mouse model will establish the foundation of knowledge for developing broadly protective vaccines against' evolutionarily divergent strains of E. chaffeensis.